PARENT SESSION

Symposium S5D Emerging techniques and systems
Wednesday September 1st, 2004 10:20 AM-12:20 PM Room 510A
Chair: Conrad Mullineaux
Co-Chair: Barry Osmond

Imaging individual chloroplasts simultaneously with third- and second-harmonic generation and multiphoton excitation fluorescence microscopy. Virginijus Barzda^*,1, Richard Cisek¹, Catherine Greenhalgh¹, Jurg Aus der Au⁴, Jeff Squier⁴, ¹ Department of Physics, Toronto, Ontario, Canada⁴ Department of Physics, Golden, Colorado, USA

ABSTRACT- Three dimensional structural investigations of chloroplasts inside a mesophyll cell in vivo was performed using non-linear femtosecond laser scanning microscopy with simultaneous detection of third harmonic generation, second harmonic generation, and multiphoton excitation fluorescence. The third harmonic was efficiently generated from the chloroplast due to the multilamelar structure of the grana and high content of carotenoids in the light-harvesting pigment-protein complexes. The second harmonic was also efficiently generated inside chloroplasts. Large portion of the second harmonic signal was originating from starch granules and the chiral arrangement of the grana. In addition, three dimensional structures of chloroplasts obtained from imaging with multiphoton excitation fluorescence of chlorophyll served as a reference for comparison with third harmonic and second harmonic generated structures. All three microscopic signals were sensitive to different physiological conditions of chloroplasts. The newly developed non-linear microscopy method is very useful for studying structural and functional dynamics of chloroplasts and other subcellular structures in vivo.

KEY WORDS: harmonic generation microscopy, light-harvesting, chloroplast structure, multiphoton excitation fluorescence